Processes of producing fermentation products

ABSTRACT

The invention relates to a process of fermenting plant material in a fermentation medium into a fermentation product using a fermenting organism, wherein one or more carbonic anhydrases are present in the fermentation medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/139,802 filed Apr. 27, 2016, now allowed, which is a division of U.S.application Ser. No. 14/246,898 filed on Apr. 7, 2014, now U.S. Pat. No.9,359,621, which is a division of U.S. application Ser. No. 13/760,140filed on Feb. 6, 2013, now U.S. Pat. No. 8,697,392, which is a divisionof U.S. application Ser. No. 13/591,572 filed on Aug. 22, 2012, now U.S.Pat. No. 8,426,160, which is a division of U.S. application Ser. No.12/682,411 filed Apr. 27, 2010, now U.S. Pat. No. 8,273,545, which is a35 U.S.C. 371 national application of PCT/US2008/080507 filed Oct. 20,2008, which claims priority or the benefit under 35 U.S.C. 119 of U.S.provisional application No. 60/980,885 filed Oct. 18, 2007, the contentsof which are fully incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to processes of fermenting plant materialinto desired fermentation products. The invention also relates toprocesses of producing a fermentation product from plant material usingone or more fermenting organisms and to compositions that can be used insuch processes.

BACKGROUND ART

Due to the limited reserves of fossil fuels and worries about emissionof greenhouse gasses there is an increasing focus on using renewableenergy sources such as plant material.

A vast number of processes of producing fermentation products,especially biofuel products such as ethanol and butanol, by fermentationof sugars derived from starch-containing and/orlignocellulose-containing material are known in the art.

However, production of such fermentation products from plant materialsis still too costly. Therefore, there is a need for providing processesthat can reduce production costs.

SUMMARY OF THE INVENTION

In the first aspect the invention relates to processes of fermentingplant material in a fermentation medium into a fermentation productusing a fermenting organism, wherein one or more carbonic anhydrases arepresent in the fermentation medium.

In the second aspect the invention relates to processes of producing afermentation product from starch-containing material comprising thesteps of:

i) liquefying starch-containing material with an alpha-amylase;

ii) saccharifying the liquefied material with a carbohydrate sourcegenerating enzyme,

iii) fermenting with one or more fermenting organisms in accordance witha fermentation process of the invention.

In the third aspect the invention relates to processes of producing afermentation product from starch-containing material, comprising thesteps of:

(a) saccharifying starch-containing material at a temperature below theinitial gelatinization temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein fermentation iscarried out in accordance with a fermentation process of the invention.

In the fourth aspect the invention relates to processes of producing afermentation product from lignocellulose-containing material, comprisingthe steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing the material;

(c) fermenting using a fermenting organism in accordance with afermentation process of the invention.

In the fifth aspect the invention relates to a composition comprisingone or more carbonic anhydrases and one or more alpha-amylases.

In the sixth aspect the invention relates to the use of a carbonicanhydrase for controlling pH fluctuation during fermentation.

In the seventh aspect the invention relates to the use of carbonicanhydrase for improving the yeast fermentation product yield and/orfermentation rate.

In the final aspect the invention relates to a transgenic plant materialtransformed with one or more carbonic anhydrase genes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of carbonic anhydrase of (CA) towardalpha-amylase (AA 1) and glucoamylase (AMG A) combo in one-stepfermentation process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes of fermenting plant materialinto a desired fermentation product. The invention also providesprocesses of producing desired fermentation products from plant materialusing a fermenting organism. Finally the invention relates to acomposition comprising one or more enzymes and one or more fermentationproduct boosting compounds.

The inventors have observed that the pH at some point in time duringyeast fermentation of plant material decreases to a pH below 4.5. Thelow pH is a drawback as some enzymes, especially alpha-amylases, undergodenaturation and/or autocleavage. The inventors have found a solution tothis problem. The concept of this invention is using carbonic anhydraseto convert CO₂, a by-product of yeast fermentation, to bicarbonate. Thechemical equation C₆H₁₂O₆→2C₂H₅OH+2CO₂ summarizes ethanol fermentation,in which one hexose molecule is converted into two ethanol molecules andtwo carbon dioxide molecules. Carbonic anhydrase can reversibly catalyzethe reaction of the by-product CO₂ and water to bicarbonate and a proton(CO₂+H₂O→HCO₃ ⁻+H⁺); a reaction that is slow at physiological pH andrequires enzyme catalysis.

Without being bound by any particular theory, the addition of carbonicanhydrase to a fermentation process is believed to serve at least twofunctions:

(1) The carbon dioxide by-product is removed from the sugar fermentationreaction equilibrium due to carbonic anhydrase catalysis and therebybalancing the reaction equilibrium towards the desired ethanol product.(2) Reduce or prevent re-conversion of bicarbonate to CO₂, thebicarbonate may react with the existing cations (potassium, calcium ormagnesium) present in the fermentation medium to bicarbonate salt andeffectively serve as buffering agent to stabilize pH fluctuation duringfermentation.

Utilizing carbonic anhydrase as a mean to maintain pH (preferably abovepH 4.5) facilitates using enzymes for producing fermentation products,such as biofuels, from starch and/or lignocellulose (i.e., biomass) withenzymes that may be less stable at low pH but are highly active onstarch or other substrates such as cellulose or hemicellulose. Moreover,maintaining pH above 4.5 will also favor fermenting organisms, such asespecially yeast which has an optimum fermentation performance at aboutpH 5.0.

In addition, bicarbonate was reported to be involved in inorganiccarboxylations of some metabolic reactions in yeast (Biochem. J. 391:311-316); in particular when urea is added as nitrogen source. Urea ismetabolized by bicarbonate-dependent urea carboxylase (J. Biol. Chem.247: 1349-1353). Thus, applying carbonic anhydrase may appropriatelyprovide the bicarbonate needed for yeast's urea metabolism.

Consequently, in the first aspect the invention relates to processes offermenting plant material in a fermentation medium into a fermentationproduct using a fermenting organism, wherein one or more carbonicanhydrases are present in the fermentation medium. The carbonicanhydrase may be added/introduced before and/or during fermentationand/or may be produced in situ by overexpression by the fermentingorganisms, preferably yeast.

According to the invention the starting material (i.e., substrate forthe fermenting organism) may be any plant material or part orconstituent thereof.

In one embodiment the stating material is starch-containing material. Inanother embodiment the starch material is lignocellulose-containingmaterial.

Fermenting Organism

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, including yeast and filamentous fungi,suitable for producing a desired fermentation product. The fermentingorganism may be C6 or C5 fermenting organisms, or a combination thereof.Both C6 and C5 fermenting organisms are well known in the art.

Especially suitable fermenting organisms according to the invention areable to ferment, i.e., convert, sugars such as glucose, fructose,maltose, xylose, mannose and/or arabinose, directly or indirectly intothe desired fermentation product.

Examples of fermenting organisms include fungal organisms such as yeast.Preferred yeast includes strains of the genus Saccharomyces, inparticular strains of Saccharomyces cerevisiae or Saccharomyces uvarum;a strain of Pichia, preferably Pichia stipitis such as Pichia stipitisCBS 5773 or Pichia pastoris; a strain of the genus Candida, inparticular a strain of Candida utilis, Candida arabinofermentans,Candida diddensii, Candida sonorensis, Candida shehatae, Candidatropicalis, or Candida boidinii. Other fermenting organisms includestrains of Hansenula, in particular Hansenula polymorpha or Hansenulaanomala; Kluyveromyces, in particular Kluyveromyces fragilis orKluyveromyces marxianus; and Schizosaccharomyces, in particularSchizosaccharomyces pombe.

Preferred bacterial fermenting organisms include strains of Escherichia,in particular Escherichia coli, strains of Zymomonas, in particularZymomonas mobilis, strains of Zymobacter, in particular Zymobactorpalmae, strains of Klebsiella in particular Klebsiella oxytoca, strainsof Leuconostoc, in particular Leuconostoc mesenteroides, strains ofClostridium, in particular Clostridium butyricum, strains ofEnterobacter, in particular Enterobacter aerogenes and strains ofThermoanaerobacter, in particular Thermoanaerobacter BG1L1 (Appl.Microbiol. Biotech. 77: 61-86) and Thermoanarobacter ethanolicus,Thermoanaerobacter thermosaccharolyticum, or Thermoanaerobactermathranii. Strains of Lactobacillus are also envisioned as are strainsof Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, andGeobacillus thermoglucosidasius.

In an embodiment the fermenting organism is a C6 sugar fermentingorganism, such as a strain of, e.g., Saccharomyces cerevisiae.

In connection with fermentation of lignocellulose derived materials, C5sugar fermenting organisms are contemplated. Most C5 sugar fermentingorganisms also ferment C6 sugars. Examples of C5 sugar fermentingorganisms include strains of Pichia, such as of the species Pichiastipitis. C5 sugar fermenting bacteria are also known. Also someSaccharomyces cerevisae strains ferment C5 (and C6) sugars. Examples aregenetically modified strains of Saccharomyces spp. that are capable offermenting C5 sugars include the ones concerned in, e.g., Ho et al.,1998, Applied and Environmental Microbiology 64(5): 1852-1859; Karhumaaet al., 2006, Microbial Cell Factories 5: 18; and Kuyper et al., 2005,FEMS Yeast Research 5: 925-934.

Certain fermenting organisms' fermentative performance may be inhibitedby the presence of inhibitors in the fermentation media and thus reduceethanol production capacity. Compounds in biomass hydrosylates and highconcentrations of ethanol are known to inhibit the fermentative capacityof certain yeast cells. Pre-adaptation or adaptation methods may reducethis inhibitory effect. Typically pre-adaptation or adaptation of yeastcells involves sequentially growing yeast cells, prior to fermentation,to increase the fermentative performance of the yeast and increaseethanol production. Methods of yeast pre-adaptation and adaptation areknown in the art. Such methods may include, for example, growing theyeast cells in the presence of crude biomass hydrolyzates; growing yeastcells in the presence of inhibitors such as phenolic compounds,furaldehydes and organic acids; growing yeast cells in the presence ofnon-inhibiting amounts of ethanol; and supplementing the yeast cultureswith acetaldehyde. In one embodiment, the fermenting organism is a yeaststrain subject to one or more pre-adaptation or adaptation methods priorto fermentation.

Certain fermenting organisms such as yeast require an adequate source ofnitrogen for propagation and fermentation. Many sources of nitrogen canbe used and such sources of nitrogen are well known in the art. In oneembodiment, a low cost source of nitrogen is used. Such low cost sourcescan be organic, such as urea, DDGs, wet cake or corn mash, or inorganic,such as ammonia or ammonium hydroxide.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

In one embodiment the fermenting organism is added to the fermentationmedium so that the viable fermenting organism, such as yeast, count permL of fermentation medium is in the range from 105 to 10¹², preferablyfrom 10⁷ to 10¹⁰, especially about 5×10⁷.

According to the invention the fermenting organism capable of producinga desired fermentation product from fermentable sugars, such as, e.g.,glucose, fructose and/or maltose, is preferably grown under preciseconditions at a particular growth rate. When the fermenting organism isintroduced into/added to the fermentation medium the inoculatedfermenting organism pass through a number of stages. Initially growthdoes not occur. This period is referred to as the “lag phase” and may beconsidered a period of adaptation. During the next phase referred to asthe “exponential phase” the growth rate gradually increases. After aperiod of maximum growth the rate ceases and the fermenting organismenters “stationary phase”. After a further period of time the fermentingorganism enters the “death phase” where the number of viable cellsdeclines.

In one embodiment the carbonic anhydrase(s) is(are) added to thefermentation medium when the fermenting organism is in lag phase.

In one embodiment the carbonic anhydrase(s) is(are) added to thefermentation medium when the fermenting organism is in exponentialphase.

In one embodiment the carbonic anhydrase is(are) added to thefermentation medium when the fermenting organism is in stationary phase.

Fermentation Products

The term “fermentation product” means a product produced by a processthat includes a fermentation step using a fermenting organism.Fermentation products contemplated according to the invention includeany fermentation product, especially biofuels products such as ethanoland butanol.

Fermentation

The fermentation may according to the invention be carried out atconventionally used conditions. Preferred fermentation processes areanaerobic processes.

For ethanol production the fermentation may in one embodiment go on for6 to 120 hours, in particular 24 to 96 hours. In an embodiment thefermentation is carried out at a temperature between 25 to 40° C.,preferably 28 to 35° C., such as 30° C. to 34° C., and in particulararound 32° C. In an embodiment the pH when initiating fermentation is inthe range from pH 3 to 6, preferably around pH 4 to 5.

Contemplated is a simultaneous hydrolysis/saccharification andfermentation (SHF/SSF) where there is no separate holding stage for thehydrolysis/saccharification, meaning that the hydrolyzing/saccharifyingenzyme(s), the fermenting organism and carbonic anhydrase(s) may beadded together. However, it should be understood that the carbonicanhydrase(s) may also be added separately. When fermentation isperformed simultaneously with hydrolysis/saccharification thetemperature is preferably between 25 to 40° C., preferably 28 to 35° C.,such as 30° C. to 34° C., in particular around 32° C., when thefermentation organism is a strain of Saccharomyces cerevisiae and thedesired fermentation product is ethanol.

Other fermentation products may be fermented at temperatures known tothe skilled person in the art to be suitable for the fermenting organismin question.

The process of the invention may be performed as a batch or as acontinuous process. The fermentation process of the invention may beconducted in an ultrafiltration system where the retentate is held underrecirculation in the presence of solids, water, and the fermentingorganism, and where the permeate is the desired fermentation productcontaining liquid. Equally contemplated if the process is conducted in acontinuous membrane reactor with ultrafiltration membranes and where theretentate is held under recirculation in presence of solids, water, thefermenting organism and where the permeate is the fermentation productcontaining liquid.

After fermentation the fermenting organism may be separated from thefermented slurry and recycled to the fermentation medium.

Recovery

Subsequent to fermentation the fermentation product may be separatedfrom the fermentation medium. The slurry may be distilled to extract thedesired fermentation product or the desired fermentation product may beextracted from the fermentation medium by micro or membrane filtrationtechniques. Alternatively the fermentation product may be recovered bystripping. Methods for recovery are well known in the art.

Production of Fermentation Products from Starch-Containing MaterialProcesses for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the present invention relates to a process for producinga fermentation product, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.

The invention relates to a process for producing a fermentation productfrom starch-containing material comprising the steps of:

i) liquefying starch-containing material with an alpha-amylase;

ii) saccharifying the liquefied material with a carbohydrate sourcegenerating enzyme,

iii) fermenting using one or more fermenting organisms, whereinfermentation is carried out in accordance with the invention, i.e., inthe presence of carbonic anhydrase.

Saccharification step ii) and fermentation step iii) may be carried outeither sequentially or simultaneously. The carbonic anhydrase may beadded before and/or during the fermentation step.

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. Suitablestarch-containing starting materials are listed in the section“Starch-containing materials” section below. Contemplated enzymes arelisted in the “Enzymes” section below. The liquefaction is preferablycarried out in the presence of an alpha-amylase, preferably a bacterialalpha-amylase or acid fungal alpha-amylase. The fermenting organism ispreferably yeast, preferably a strain of Saccharomyces. Suitablefermenting organisms are listed in the “Fermenting Organisms” sectionabove.

In a particular embodiment, the process of the invention furthercomprises, prior to the step (i), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling; y) forming a slurry comprising thestarch-containing material and water.

The aqueous slurry may contain from 10-55 wt. % dry solids, preferably25-45 wt. % dry solids (DS), more preferably 30-40 wt. % dry solids ofstarch-containing material. The slurry is heated to above thegelatinization temperature and alpha-amylase, preferably bacterialand/or acid fungal alpha-amylase may be added to initiate liquefaction(thinning). The slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to an alpha-amylase in step(i) of the invention.

More specifically liquefaction may be carried out as a three-step hotslurry process. The slurry is heated to between 60-95° C., preferably80-85° C., and alpha-amylase is added to initiate liquefaction(thinning). Then the slurry may be jet-cooked at a temperature between95-140° C., preferably 105-125° C., for 1-15 minutes, preferably for3-10 minutes, especially around 5 minutes. The slurry is cooled to60-95° C. and more alpha-amylase is added to finalize hydrolysis(secondary liquefaction). The liquefaction process is usually carriedout at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled andliquefied whole grains are known as mash.

The saccharification in step (ii) may be carried out using conditionswell know in the art. For instance, a full saccharification process maylast up to from about 24 to about 72 hours, however, it is common onlyto do a pre-saccharification of typically 40-90 minutes at a temperaturebetween 30-65° C., typically about 60° C., followed by completesaccharification during fermentation in a simultaneous saccharificationand fermentation process (SSF process). Saccharification is typicallycarried out at temperatures from 20-75° C., preferably from 40-70° C.,typically around 60° C., and at a pH between 4 and 5, normally at aboutpH 4.5.

The most widely used process in fermentation product, especiallyethanol, production is the simultaneous saccharification andfermentation (SSF) process, in which there is no holding stage for thesaccharification, meaning that fermenting organism, such as yeast, andenzyme(s) may be added together. SSF may typically be carried out at atemperature between 25° C. and 40° C., such as between 28° C. and 35°C., such as between 30° C. and 34° C., preferably around 32° C.According to the invention the temperature may be adjusted up or downduring fermentation.

In accordance with the present invention the fermentation step (iii)includes, without limitation, fermentation processes of the inventionused to produce fermentation products as exemplified above in the“Fermentation Products” section.

Processes for Producing Fermentation Products from Un-GelatinizedStarch-Containing

In this aspect the invention relates to processes for producing afermentation product from starch-containing material withoutgelatinization (often referred to as “cooking”) of the starch-containingmaterial. According to the invention the desired fermentation product,such as ethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material. In one embodiment a processof the invention includes saccharifying (e.g., milled) starch-containingmaterial, e.g., granular starch, below the initial gelatinizationtemperature, preferably in the presence of an alpha-amylase and/or ancarbohydrate-source generating enzyme to produce sugars that can befermented into the desired fermentation product by a suitable fermentingorganism.

In this embodiment the desired fermentation product, preferably ethanol,is produced from ungelatinized (i.e., uncooked), preferably milled corn.

Accordingly, in this aspect the invention relates to a process ofproducing a fermentation product from starch-containing material,comprising the steps of:

(a) saccharifying starch-containing material at a temperature below theinitial gelatinization temperature of said starch-containing material,

(b) fermenting using a fermenting organism, wherein the fermentation iscarried out in accordance with the fermentation process of theinvention, i.e., in the presence of carbonic anhydrase.

In a preferred embodiment steps (a) and (b) are carried outsimultaneously (i.e., one step fermentation) or sequentially.

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. Suitablestarch-containing starting materials are listed in the section“Starch-Containing materials” section below. Contemplated enzymes arelisted in the “Enzymes” section below. The alpha-amylase used ispreferably an acid alpha-amylase, preferably acid fungal alpha-amylase.The fermenting organism is preferably yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”-section above.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. In generalstarch heated in water begins to gelatinize between 50° C. and 75° C.;the exact temperature of gelatinization depends on the specific starchand can readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Stärke 44(12): 461-466.

Before step (a) a slurry of starch-containing material, such as granularstarch, having 10-55 wt. % dry solids (DS), preferably 25-45 wt. % drysolids, more preferably 30-40 wt. % dry solids of starch-containingmaterial may be prepared. The slurry may include water and/or processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side-stripper water from distillation, orprocess water from other fermentation product plants. Because theprocess of the invention is carried out below the gelatinizationtemperature and thus no significant viscosity increase takes place, highlevels of stillage may be used if desired. In an embodiment the aqueousslurry contains from about 1 to about 70 vol. % stillage, preferably15-60% vol. % stillage, especially from about 30 to 50 vol. % stillage.

The starch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material is convertedinto a soluble starch hydrolysate.

A process of the invention is conducted at a temperature below theinitial gelatinization temperature, which means that the temperature atwhich step (a) is carried out typically lies in the range between 30-75°C., preferably between 45-60° C.

In a preferred embodiment step (a) and step (b) are carried out as asimultaneous saccharification and fermentation process. In suchpreferred embodiment the process is typically carried at a temperaturebetween 25° C. and 40° C., such as between 28° C. and 35° C., such asbetween 30° C. and 34° C., preferably around 32° C.

In an embodiment simultaneous saccharification and fermentation iscarried out so that the sugar level, such as glucose level, is kept at alow level such as below 6 wt. %, preferably below about 3 wt. %,preferably below about 2 wt. %, more preferred below about 1 wt. %, evenmore preferred below about 0.5 wt. %, or even more preferred 0.25 wt. %,such as below about 0.1 wt. %. Such low levels of sugar can beaccomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich quantities of enzyme and fermenting organism to use. The employedquantities of enzyme and fermenting organism may also be selected tomaintain low concentrations of maltose in the fermentation broth. Forinstance, the maltose level may be kept below about 0.5 wt. % or belowabout 0.2 wt. %.

The process of the invention may be carried out at a pH in the rangebetween 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH4 to 5.

Starch-Containing materials

Any suitable starch-containing starting material, including granularstarch, may be used according to the present invention. The startingmaterial is generally selected based on the desired fermentationproduct. Examples of starch-containing starting materials, suitable foruse in a process of present invention, include tubers, roots, stems,whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava,tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixturesthereof, or cereals. Contemplated are also waxy and non-waxy types ofcorn and barley.

The term “granular starch” means uncooked starch, i.e., starch in itsnatural form found in cereal, tubers or grains. Starch is formed withinplant cells as tiny granules insoluble in water. When put in cold water,the starch granules may absorb a small amount of the liquid and swell.At temperatures up to 50° C. to 75° C. the swelling may be reversible.However, with higher temperatures an irreversible swelling called“gelatinization” begins. Granular starch to be processed may be a highlyrefined starch quality, preferably at least 90%, at least 95%, at least97% or at least 99.5% pure or it may be a more crude starch-containingmaterials comprising milled whole grain including non-starch fractionssuch as germ residues and fibers. The raw material, such as whole grain,is reduced in particle size, e.g., by milling, in order to open up thestructure and allowing for further processing. Two processes arepreferred according to the invention: wet and dry milling. In drymilling whole kernels are milled and used. Wet milling gives a goodseparation of germ and meal (starch granules and protein) and is oftenapplied at locations where the starch hydrolysate is used in productionof syrups. Both dry and wet milling is well known in the art of starchprocessing and is equally contemplated for the process of the invention.In an embodiment the particle size is reduced to between 0.05 to 3.0 mm,preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%,more preferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen.

Production of Fermentation Products from Lignocellulose-ContainingMaterial

In this aspect, the invention relates to processes of producingfermentation products from lignocellulose-containing material.Conversion of lignocellulose-containing material into fermentationproducts, such as ethanol, has the advantages of the ready availabilityof large amounts of feedstock, including wood, agricultural residues,herbaceous crops, municipal solid wastes etc. Lignocellulose-containingmaterials primarily consist of cellulose, hemicellulose, and lignin andare often referred to as “biomass”.

The structure of lignocellulose is not directly accessible to enzymatichydrolysis. Therefore, the lignocellulose-containing material has to bepre-treated, e.g., by acid hydrolysis under adequate conditions ofpressure and temperature, in order to break the lignin seal and disruptthe crystalline structure of cellulose. This causes solubilization ofthe hemicellulose and cellulose fractions. The cellulose andhemicelluloses can then be hydrolyzed enzymatically, e.g., bycellulolytic enzymes, to convert the carbohydrate polymers intofermentable sugars which may be fermented into desired fermentationproducts, such as ethanol. Optionally the fermentation product may berecovered, e.g., by distillation.

In this aspect the invention relates to a process of producing afermentation product from lignocellulose-containing material, comprisingthe steps of:

(a) pre-treating lignocellulose-containing material;

(b) hydrolyzing the material;

(c) fermenting with a fermenting organism in accordance with thefermentation process of the invention, i.e., in the presence of carbonicanhydrase.

The carbonic anhydrase may be added before and/or during fermentation.Hydrolysis steps (b) and fermentation step (c) may be carried outsequentially or simultaneously. In preferred embodiments the steps arecarried out as SHF or HHF process steps which will be described furtherbelow.

SSF, HHF and SHF

Hydrolysis and fermentation can be carried out as a simultaneoushydrolysis and fermentation step (SSF). In general this means thatcombined/simultaneous hydrolysis and fermentation are carried out atconditions (e.g., temperature and/or pH) suitable, preferably optimal,for the fermenting organism(s) in question.

Hydrolysis and fermentation can also be carried out as hybrid hydrolysisand fermentation (HHF). HHF typically begins with a separate partialhydrolysis step and ends with a simultaneous hydrolysis and fermentationstep. The separate partial hydrolysis step is an enzymatic cellulosesaccharification step typically carried out at conditions (e.g., athigher temperatures) suitable, preferably optimal, for the hydrolyzingenzyme(s) in question. The subsequent simultaneous hydrolysis andfermentation step is typically carried out at conditions suitable forthe fermenting organism(s) (often at lower temperatures than theseparate hydrolysis step).

Hydrolysis and fermentation can also be carried out as separatehydrolysis and fermentation, where the hydrolysis is taken to completionbefore initiation of fermentation. This is often referred to as “SHF”.

Pre-Treatment

The lignocellulose-containing material may according to the invention bepre-treated before being hydrolyzed and/or fermented. In a preferredembodiment the pre-treated material is hydrolyzed, preferablyenzymatically, before and/or during fermentation. The goal ofpre-treatment is to separate and/or release cellulose, hemicelluloseand/or lignin and this way improve the rate of enzymatic hydrolysis.

According to the invention pre-treatment step (a) may be a conventionalpre-treatment step known in the art. Pre-treatment may take place inaqueous slurry. The lignocellulose-containing material may duringpre-treatment be present in an amount between 10-80 wt. %, preferablybetween 20-50 wt. %.

Chemical, Mechanical and/or Biological Pre-Treatment

The lignocellulose-containing material may according to the invention bechemically, mechanically and/or biologically pre-treated beforehydrolysis and/or fermentation. Mechanical treatment (often referred toas physical pre-treatment) may be used alone or in combination withsubsequent or simultaneous hydrolysis, especially enzymatic hydrolysis,to promote the separation and/or release of cellulose, hemicelluloseand/or lignin.

Preferably, the chemical, mechanical and/or biological pre-treatment iscarried out prior to the hydrolysis and/or fermentation. Alternatively,the chemical, mechanical and/or biological pre-treatment is carried outsimultaneously with hydrolysis, such as simultaneously with addition ofone or more cellulolytic enzymes, or other enzyme activities mentionedbelow, to release fermentable sugars, such as glucose and/or maltose.

In an embodiment of the invention the pre-treatedlignocellulose-containing material is washed and/or detoxified beforehydrolysis step (b). This may improve the fermentability of, e.g.,dilute-acid hydrolyzed lignocellulose-containing material, such as cornstover. Detoxification may be carried out in any suitable way, e.g., bysteam stripping, evaporation, ion exchange, resin or charcoal treatmentof the liquid fraction or by washing the pre-treated material.

Chemical Pre-Treatment

According to the present invention “chemical pre-treatment” refers toany chemical treatment which promotes the separation and/or release ofcellulose, hemicellulose and/or lignin. Examples of suitable chemicalpre-treatment steps include treatment with; for example, dilute acid,lime, alkaline, organic solvent, ammonia, sulfur dioxide, carbondioxide. Further, wet oxidation and pH-controlled hydrothermolysis arealso contemplated chemical pre-treatments.

Preferably, the chemical pre-treatment is acid treatment, morepreferably, a continuous dilute and/or mild acid treatment, such as,treatment with sulfuric acid, or another organic acid, such as aceticacid, citric acid, tartaric acid, succinic acid, or mixtures thereof.Other acids may also be used. Mild acid treatment means in the contextof the present invention that the treatment pH lies in the range from1-5, preferably 1-3. In a specific embodiment the acid concentration isin the range from 0.1 to 2.0 wt. % acid, preferably sulphuric acid. Theacid may be mixed or contacted with the material to be fermentedaccording to the invention and the mixture may be held at a temperaturein the range of 160-220° C., such as 165-195° C., for periods rangingfrom minutes to seconds, e.g., 1-60 minutes, such as 2-30 minutes or3-12 minutes. Addition of strong acids, such as sulphuric acid, may beapplied to remove hemicellulose. This enhances the digestibility ofcellulose.

Cellulose solvent treatment, also contemplated according to theinvention, has been shown to convert about 90% of cellulose to glucose.It has also been shown that enzymatic hydrolysis could be greatlyenhanced when the lignocellulosic structure is disrupted. Alkaline H₂O₂,ozone, organosolv (uses Lewis acids, FeCl₃, (Al)₂SO₄ in aqueousalcohols), glycerol, dioxane, phenol, or ethylene glycol are amongsolvents known to disrupt cellulose structure and promote hydrolysis(Mosier et al., 2005, Bioresource Technology 96: 673-686).

Alkaline chemical pre-treatment with base, e.g., NaOH, Na₂CO₃ and/orammonia or the like, is also within the scope of the invention.Pre-treatment methods using ammonia are described in, e.g., WO2006/110891, WO 2006/110899, WO 2006/110900, WO 2006/110901, which arehereby incorporated by reference.

Wet oxidation techniques involve use of oxidizing agents, such as:sulphite based oxidizing agents or the like. Examples of solventpre-treatments include treatment with DMSO (Dimethyl Sulfoxide) or thelike. Chemical pre-treatment is generally carried out for 1 to 60minutes, such as from 5 to 30 minutes, but may be carried out forshorter or longer periods of time dependent on the material to bepre-treated.

Other examples of suitable pre-treatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechn. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and US publication no.2002/0164730, which references are hereby all incorporated by reference.

Mechanical Pre-Treatment

As used in context of the present invention the term “mechanicalpre-treatment” refers to any mechanical or physical pretreatment whichpromotes the separation and/or release of cellulose, hemicelluloseand/or lignin from lignocellulose-containing material. For example,mechanical pre-treatment includes various types of milling, irradiation,steaming/steam explosion, and hydrothermolysis.

Mechanical pre-treatment includes comminution (mechanical reduction ofthe particle size). Comminution includes dry milling, wet milling andvibratory ball milling. Mechanical pre-treatment may involve highpressure and/or high temperature (steam explosion). In an embodiment ofthe invention high pressure means pressure in the range from 300 to 600psi, preferably 400 to 500 psi, such as around 450 psi. In an embodimentof the invention high temperature means temperatures in the range fromabout 100 to 300° C., preferably from about 140 to 235° C. In apreferred embodiment mechanical pre-treatment is a batch-process, steamgun hydrolyzer system which uses high pressure and high temperature asdefined above. A Sunds Hydrolyzer (available from Sunds Defibrator AB(Sweden) may be used for this.

Combined Chemical and Mechanical Pretreatment

In an embodiment of the invention both chemical and mechanicalpre-treatments are carried out involving, for example, both dilute ormild acid pretreatment and high temperature and pressure treatment. Thechemical and mechanical pretreatment may be carried out sequentially orsimultaneously, as desired.

Accordingly, in a preferred embodiment, the lignocellulose-containingmaterial is subjected to both chemical and mechanical pretreatment topromote the separation and/or release of cellulose, hemicellulose and/orlignin.

In a preferred embodiment the pre-treatment is carried out as a diluteand/or mild acid steam explosion step. In another preferred embodimentpre-treatment is carried out as an ammonia fiber explosion step (or AFEXpretreatment step).

Biological Pre-Treatment

As used in the present invention the term “biological pretreatment”refers to any biological pretreatment which promotes the separationand/or release of cellulose, hemicellulose, and/or lignin from thelignocellulose-containing material. Biological pre-treatment techniquescan involve applying lignin-solubilizing microorganisms (see, forexample, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993,Physicochemical and biological treatments for enzymatic/microbialconversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39:295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: areview, in Enzymatic Conversion of Biomass for Fuels Production, Himmel,M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566,American Chemical Society, Washington, D.C., chapter 15; Gong, C. S.,Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Hydrolysis

Before and/or during fermentation the pre-treatedlignocellulose-containing material may be hydrolyzed in order to breakthe lignin seal and disrupt the crystalline structure of cellulose. In apreferred embodiment hydrolysis is carried out enzymatically. Accordingto the invention the pre-treated lignocellulose-containing material tobe fermented may be hydrolyzed by one or more hydrolases (class EC 3according to Enzyme Nomenclature), preferably one or more carbohydrasesincluding cellulolytic enzymes and hemicellulolytic enzymes, orcombinations thereof. Further, protease, alpha-amylase, glucoamylaseand/or the like may also be present during hydrolysis and/orfermentation as the lignocellulose-containing material may include some,e.g., starchy and/or proteinaceous material.

The enzyme(s) used for hydrolysis is(are) capable of directly orindirectly converting carbohydrate polymers into fermentable sugars,such as glucose and/or maltose, which can be fermented into a desiredfermentation product, such as ethanol.

In a preferred embodiment the carbohydrase(s) has(have) cellulolyticand/or hemicellulolytic enzyme activity.

In a preferred embodiment hydrolysis is carried out using a cellulolyticenzyme preparation further comprising one or more polypeptides havingcellulolytic enhancing activity.

In a preferred embodiment the polypeptide(s) having cellulolyticenhancing activity is(are) of family GH61A origin. Examples of suitableand preferred cellulolytic enzyme preparations and polypeptides havingcellulolytic enhancing activity are described in the “CellulolyticEnzymes” section and “Cellulolytic Enhancing polypeptides” sectionbelow.

Suitable enzymes are described in the “Enzymes” section below.

Hemicellulose polymers can be broken down by hemicellullolytic enzymesand/or acid hydrolysis to release its five and six carbon sugarcomponents. The six carbon sugars (hexoses), such as glucose, galactose,arabinose, and mannose, can readily be fermented to fermentationproducts such as ethanol, acetone, butanol, glycerol, citric acid,fumaric acid etc. by suitable fermenting organisms including yeast.

Yeast is the preferred fermenting organism for ethanol fermentation.Preferred are strains of Saccharomyces, especially strains of thespecies Saccharomyces cerevisiae, preferably strains which are resistanttowards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or20 vol. % or more ethanol.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions which can readily be determined by oneskilled in the art. In a preferred embodiment hydrolysis is carried outat suitable, preferably optimal, conditions for the enzyme(s) inquestion.

Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. Preferably, hydrolysis is carriedout at a temperature between 25 and 70° C., preferably between 40 and60° C., especially around 50° C. The step is preferably carried out at apH in the range from 3-8, preferably pH 4-6. Hydrolysis is typicallycarried out for between 12 and 96 hours, preferable 16 to 72 hours, morepreferably between 24 and 48 hours.

Lignocellulose-Containing Material (Biomass)

Any suitable lignocellulose-containing material is contemplated incontext of the present invention. Lignocellulose-containing material maybe any material containing lignocellulose. In a preferred embodiment thelignocellulose-containing material contains at least 50 wt. %,preferably at least 70 wt. %, more preferably at least 90 wt. %lignocellulose. It is to be understood that thelignocellulose-containing material may also comprise other constituentssuch as cellulosic material, such as cellulose, hemicellulose and mayalso comprise constituents such as sugars, such as fermentable sugarsand/or un-fermentable sugars.

Ligno-cellulose-containing material is generally found, for example, inthe stems, leaves, hulls, husks, and cobs of plants or leaves, branches,and wood of trees. Lignocellulosic material can also be, but is notlimited to, herbaceous material, agricultural residues, forestryresidues, municipal solid wastes, waste paper, and pulp and paper millresidues. It is understood herein that lignocellulose-containingmaterial may be in the form of plant cell wall material containinglignin, cellulose, and hemi-cellulose in a mixed matrix.

In one embodiment the lignocellulose-containing material is selectedfrom one or more of corn fiber, rice straw, pine wood, wood chips,poplar, bagasse, and paper and pulp processing waste.

Other examples of suitable lignocellulose-containing material includecorn stover, corn cobs, hard wood such as poplar and birch, soft wood,cereal straw such as wheat straw, switch grass, Miscanthus, rice hulls,municipal solid waste (MSW), industrial organic waste, office paper, ormixtures thereof.

In a preferred embodiment the lignocellulose-containing material is cornstover or corn cobs. In another preferred embodiment, thelignocellulose-containing material is corn fiber. In another preferredembodiment, the lignocellulose-containing material is switch grass. Inanother preferred embodiment, the lignocellulose-containing material isbagasse.

Enzymes

Even if not specifically mentioned in context of a process of theinvention, it is to be understood that the enzyme(s) is(are) used in an“effective amount”.

Carbonic Anhydrase

According to the invention any carbonic anhydrase (CA) may be presentduring fermentation. In a preferred embodiment the carbonic anhydrase isof microbial origin, such as bacterial or fungal, such as yeast orfilamentous fungus origin. In another embodiment the carbonic anhydraseis of mammalian or plant origin.

Carbonic anhydrases (also termed carbonate dehydratases) catalyze theinter-conversion between carbon dioxide and bicarbonate [CO₂+H₂O⇄HCO₃⁻+H⁺]. An example of a carbonic anhydrase (CA) includes the onediscovered in bovine blood (Meldrum and Roughton, 1933, J. Physiol. 80:113-142). Anhydrases are categorized in three distinct classes calledthe alpha-, beta- and gamma-class, and potentially a fourth class, thedelta-class (Bacteria, Archaea, Eukarya; Tripp et al., 2001, J. Biol.Chem. 276: 48615-48618). For alpha-Cas more than 11 isozymes have beenidentified in mammals. Alpha-carbonic anhydrases are abundant in allmammalian tissues where they facilitate the removal of CO₂. Beta-Cas areubiquitous in algae and plants where they provide for CO₂ uptake andfixation for photosynthesis. Gamma-Cas include one from ArchaeonMethanosarcina thermophila strain TM-1 (Alber and Ferry, 1994, Proc.Natl. Acad. Sci. USA 91: 6909-6913) and the ones disclosed by Parisi etal., 2004, Plant Mol. Biol. 55: 193-207. In prokaryotes genes encodingall three CA classes have been identified, with the beta- andgamma-class predominating. Many prokaryotes contain carbonic anhydrasegenes from more than one class or several genes of the same class (forreview see Smith and Ferry, 2000, FEMS Microbiol. Rev. 24: 335-366;Tripp et al., 2001, J. Biol. Chem. 276: 48615-48618.

Mammalian-, plant- and prokaryotic carbonic anhydrases (alpha- andbeta-class carbonis anhydrases) generally function at physiologicaltemperatures (37° C.) or lower temperatures.

In another embodiment the carbonic anhydrase is the beta-class CA (Cab)from Methanobacterium thermoautotrophicum ΔH (Smith and Ferry, 1999, J.Bacteriol. 181: 6247-6253) or the gamma-class carbonic anhydrase (Cam)from Methanosarcina thermophilaTM-1 (Alber and Ferry, 1994, Proc. Natl.Acad. Sci. USA 91: 6909-6913; Alber and Ferry, 1996, J. Bacteriol. 178:3270-3274).

Other examples of carbonic anhydrases include carbonic anhydrasesdisclosed as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10 or SEQ ID NO: 12 from Bacillus clausii or from Bacillusclausii KSM-K16 (NCBI acc. No. Q5WD44 or SEQ ID NO: 14) or from Bacillushalodurans (NCBI acc. No. Q9KFW1 or SEQ ID NO: 16) all disclosed inco-pending U.S. provisional application No. 60/887,386 from Novozymes,which is incorporated by reference.

In a preferred embodiment the carbonic anhydrase is derived fromBacillus sp. P203 deposited under accession # DSM 19153. The Bacillussp. P203 carbonic anhydrase is disclosed and concerned in SEQ ID NO: 4and Examples 8-10 in WO 2007/019859 (Novozymes A/S) which is herebyincorporated by reference.

According to a preferred embodiment of the invention the carbonicanhydrase is present or added to the fermentation medium in aconcentration of 100-100,000 Units/g dry solids (DS), preferably1,000-20,000 Units/g DS.

Alpha-Amylase

According to the invention any alpha-amylase may be used. In a preferredembodiment the alpha-amylase is an acid alpha-amylase, e.g., acid fungalalpha-amylase or acid bacterial alpha-amylase. The term “acidalpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which added in aneffective amount has activity optimum at a pH in the range of 3 to 7,preferably from 3.5 to 6, or more preferably from 4-5.

In one embodiment an alpha-amylase is present before and/or duringfermentation. In a preferred embodiment the alpha-amylase is analpha-amylase that has less than 70%, preferably less than 60%, morepreferably less than 50%, even more preferably less than 40%, morepreferably less than 30% residual activity left at a pH below 6,preferably pH 5, especially below pH 4.5 compared to its maximumactivity (i.e., 100%). The residual activity of an alpha-amylase may betested using the pH stability assay described in the “Materials &Methods” section.

Bacterial Alpha-Amylase

According to the invention the bacterial alpha-amylase is preferablyderived from the genus Bacillus.

In a preferred embodiment the Bacillus alpha-amylase is derived from astrain of Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillussubtilis or Bacillus stearothermophilus, but may also be derived fromother Bacillus sp. Specific examples of contemplated alpha-amylasesinclude the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase SEQ ID NO:5 in WO 99/19467 and the Bacillus stearothermophilus alpha-amylase shownin SEQ ID NO: 3 in WO 99/19467 (all sequences hereby incorporated byreference). In an embodiment the alpha-amylase may be an enzyme having adegree of identity of at least 60%, preferably at least 70%, morepreferred at least 80%, even more preferred at least 90%, such as atleast 95%, at least 96%, at least 97%, at least 98% or at least 99% toany of the sequences shown in SEQ ID NO: 1, 2 or 3, respectively, in WO99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562,6,297,038 or U.S. Pat. No. 6,187,576 (hereby incorporated by reference)and include Bacillus stearothermophilus alpha-amylase (BSGalpha-amylase) variants having a deletion of one or two amino acid inpositions R179 to G182, preferably a double deletion disclosed in WO96/23873—see, e.g., page 20, lines 1-10 (hereby incorporated byreference), preferably corresponding to delta(181-182) compared to thewild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3disclosed in WO 99/19467 or deletion of amino acids R179 and G180 usingSEQ ID NO:3 in WO 99/19467 for numbering (which reference is herebyincorporated by reference). Even more preferred are Bacillusalpha-amylases, especially Bacillus stearothermophilus alpha-amylase,which have a double deletion corresponding to delta(181-182) and furthercomprise a N193F substitution (also denoted 1181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO:3 disclosed in WO 99/19467.

Bacterial Hybrid Alpha-Amylase

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), withone or more, especially all, of the following substitution:G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferredare variants having one or more of the following mutations (orcorresponding mutations in other Bacillus alpha-amylase backbones):H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residuesbetween positions 176 and 179, preferably deletion of E178 and G179(using the SEQ ID NO: 5 numbering of WO 99/19467).

In an embodiment the bacterial alpha-amylase is dosed in an amount of0.0005-5 KNU per g DS, preferably 0.001-1 KNU per g DS, such as around0.050 KNU per g DS.

Fungal Alpha-Amylase

Fungal alpha-amylases include alpha-amylases derived from a strain ofthe genus Aspergillus, such as, Aspergillus oryzae, Aspergillus nigerand Aspergillus kawachii alpha-amylases.

A preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is derived from a strain of Aspergillus oryzae. According to thepresent invention, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is the one from Aspergillus niger disclosed as“AMYA_ASPNG” in the Swiss-prot/TeEMBL database under the primaryaccession no. P56271 and described in WO 89/01969 (Example3—incorporated by reference). A commercially available acid fungalalpha-amylase derived from Aspergillus niger is SP288 (available fromNovozymes A/S, Denmark).

Other contemplated wild-type alpha-amylases include those derived from astrain of the genera Rhizomucor and Meripilus, preferably a strain ofRhizomucor pusillus (WO 2004/055178 incorporated by reference) orMeripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81:292-298, “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii.”; and further as EMBL:#AB008370.

The fungal alpha-amylase may also be a wild-type enzyme comprising astarch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e.,none-hybrid), or a variant thereof. In an embodiment the wild-typealpha-amylase is derived from a strain of Aspergillus kawachii.

Fungal Hybrid Alpha-Amylase

In a preferred embodiment the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. Patent Publicationno. 2005/0054071 (Novozymes) or U.S. patent application No. 60/638,614(Novozymes) which is hereby incorporated by reference. A hybridalpha-amylase may comprise an alpha-amylase catalytic domain (CD) and acarbohydrate-binding domain/module (CBM), such as a starch bindingdomain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in Table 1 to 5 of the examples in U.S. patent application No.60/638,614, including Fungamyl variant with catalytic domain JA118 andAthelia rolfsii SBD (SEQ ID NO:100 in U.S. 60/638,614), Rhizomucorpusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ IDNO:101 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase withAspergillus niger glucoamylase linker and SBD (which is disclosed inTable 5 as a combination of amino acid sequences SEQ ID NO:20, SEQ IDNO:72 and SEQ ID NO:96 in U.S. application Ser. No. 11/316,535) or asV039 in Table 5 in WO 2006/069290, and Meripilus giganteus alpha-amylasewith Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO:102 in U.S.60/638,614). Other specifically contemplated hybrid alpha-amylases areany of the ones listed in Tables 3, 4, 5, and 6 in Example 4 in U.S.application Ser. No. 11/316,535 and WO 2006/069290 (hereby incorporatedby reference).

Other specific examples of contemplated hybrid alpha-amylases includethose disclosed in U.S. Patent Publication no. 2005/0054071, includingthose disclosed in Table 3 on page 15, such as Aspergillus nigeralpha-amylase with Aspergillus kawachii linker and starch bindingdomain.

Contemplated are also alpha-amylases which exhibit a high identity toany of above mention alpha-amylases, i.e., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or even 100% identity to themature enzyme sequences.

An acid alpha-amylases may according to the invention be added in anamount of 0.001 to 10 AFAU/g DS, preferably from 0.01 to 5 AFAU/g DS,especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01to 1 FAU-F/g DS.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE™ from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™,LIQUOZYME™ X, LIQUOZYME™ SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S)and CLARASE™ L-40,000, DEX-LO™, SPEZYME® FRED-L, SPEZYME® HPA, SPEZYME®ALPHA, SPEZYME® XTRA, SPEZYME® AA, SPEZYME® DELTA AA, and GC358(Genencor Int.), FUELZYME™-LF (Verenium Inc), and the acid fungalalpha-amylase sold under the trade name SP288 (available from NovozymesA/S, Denmark).

Carbohydrate-Source Generating Enzyme

The term “carbohydrate-source generating enzyme” includes glucoamylase(being glucose generators), beta-amylase and maltogenic amylase (beingmaltose generators) and also pullulanase and alpha-glucosidase. Acarbohydrate-source generating enzyme is capable of producing acarbohydrate that can be used as an energy-source by the fermentingorganism(s) in question, for instance, when used in a process of theinvention for producing a fermentation product, such as ethanol. Thegenerated carbohydrate may be converted directly or indirectly to thedesired fermentation product, preferably ethanol. According to theinvention a mixture of carbohydrate-source generating enzymes may beused. Especially contemplated mixtures are mixtures of at least aglucoamylase and an alpha-amylase, especially an acid amylase, even morepreferred an acid fungal alpha-amylase. The ratio between acid fungalalpha-amylase activity (AFAU) and glucoamylase activity (AGU) (i.e.,AFAU per AGU) may in an embodiment of the invention be between 0.1 and100, in particular between 2 and 50, such as in the range from 10-40.

Glucoamylase

A glucoamylase used according to the invention may be derived from anysuitable source, e.g., derived from a microorganism or a plant.Preferred glucoamylases are of fungal or bacterial origin, selected fromthe group consisting of Aspergillus glucoamylases, in particularAspergillus niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J.3(5): 1097-1102), or variants thereof, such as those disclosed in WO92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A.awamori glucoamylase disclosed in WO 84/02921, Aspergillus oryzaeglucoamylase (Agric. Biol. Chem. 55(4): 941-949 (1991)), or variants orfragments thereof. Other Aspergillus glucoamylase variants includevariants with enhanced thermal stability: G137A and G139A (Chen et al.,1996, Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995,Prot. Eng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301:275-281); disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry35: 8698-8704; and introduction of Pro residues in position A435 andS436 (Li et al., 1997, Protein Eng. 10: 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al., 1998, “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, Appl.Microbiol. Biotechnol. 50: 323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215).

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831) and Trametes cingulata,Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed inWO 2006/069289; or Peniphora rufomarginata disclosed inPCT/US2007/066618; or a mixture thereof. Also hybrid glucoamylase arecontemplated according to the invention. Examples the hybridglucoamylases disclosed in WO 2005/045018. Specific examples include thehybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (whichhybrids are hereby incorporated by reference).

Contemplated are also glucoamylases which exhibit a high identity to anyof above mention glucoamylases, i.e., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or even 100% identity to themature enzymes sequences mentioned above.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA and AMG™ E (from Novozymes A/S);OPTIDEX™ 300, GC480, GC147 (from Genencor Int.); AMIGASE™ and AMIGASE™PLUS (from DSM); G-ZYME® G900, G-ZYME®, G-ZYME® 480 ETHANOL, DISTILLASE®L-400, DISTILLASE® L-500, DISTILLASE® VHP, and G990 ZR (from GenencorInt.).

Glucoamylases may in an embodiment be added in an amount of 0.0001-20AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/gDS, such as 0.1-2 AGU/g DS.

Beta-Amylase

A beta-amylase (E.C 3.2.1.2) is the name traditionally given toexo-acting maltogenic amylases, which catalyze the hydrolysis of1,4-alpha-glucosidic linkages in amylose, amylopectin and relatedglucose polymers. Maltose units are successively removed from thenon-reducing chain ends in a step-wise manner until the molecule isdegraded or, in the case of amylopectin, until a branch point isreached. The maltose released has the beta anomeric configuration, hencethe name beta-amylase.

Beta-amylases have been isolated from various plants and microorganisms(Fogarty and Kelly, 1979, Progress in Industrial Microbiology 15:112-115). These beta-amylases are characterized by having optimumtemperatures in the range from 40° C. to 65° C. and optimum pH in therange from 4.5 to 7. A commercially available beta-amylase from barleyis NOVOZYM™ WBA from Novozymes A/S, Denmark and SPEZYME™ BBA 1500 fromGenencor Int., USA.

Maltogenic Amylase

The amylase may also be a maltogenic alpha-amylase. A “maltogenicalpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is ableto hydrolyze amylose and amylopectin to maltose in thealpha-configuration. A maltogenic amylase from Bacillusstearothermophilus strain NCIB 11837 is commercially available fromNovozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat. Nos.4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated byreference.

The maltogenic amylase may in a preferred embodiment be added in anamount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.

The carbohydrate-source generating enzyme may be any carbohydrate-sourcegenerating enzyme, including the ones listed in the “Carbohydrate-SourceGenerating Enzymes” section above. In a preferred embodiment thecarbohydrate-source generating enzyme is a glucoamylase. In an preferredembodiment the glucoamylase is selected from the group derived from astrain of Aspergillus, preferably Aspergillus niger or Aspergillusawamori, a strain of Talaromyces, especially Talaromyces emersonii; or astrain of Athelia, especially Athelia rolfsii; a strain of Trametes,preferably Trametes cingulata; a strain of the genus Pachykytospora,preferably a strain of Pachykytospora papyracea; or a strain of thegenus Leucopaxillus, preferably Leucopaxillus giganteus; or a strain ofthe genus Peniophora, preferably a strain of the species Peniophorarufomarginata; or a mixture thereof.

The alpha-amylase may be any alpha-amylase, including the ones mentionedin the “Alpha-Amylases” section above. In a preferred embodiment thealpha-amylase is an acid alpha-amylase, especially an acid fungalalpha-amylase. In a preferred embodiment the alpha-amylase is selectedfrom the group of fungal alpha-amylases. In a preferred embodiment thealpha-amylase is derived from the genus Aspergillus, especially a strainof A. niger, A. oryzae, A. awamori, or Aspergillus kawachii, or of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, or thegenus Meripilus, preferably a strain of Meripilus giganteus.

The ratio between glucoamylase activity (AGU) and fungal alpha-amylaseactivity (FAU-F) (the ratio of AGU per FAU-F) may in an embodiment ofthe invention be between 0.1 and 100 AGU/FAU-F, in particular between 2and 50 AGU/FAU-F, such as in the range from 10-40 AGU/FAU-F. The ratioof acid alpha-amylase to glucoamylase is in the range between 0.3 and5.0 AFAU/AGU. Above composition of the invention is suitable for use ina process for producing fermentation products, such as ethanol, of theinvention.

Cellulolytic Activity

The term “cellulolytic activity” as used herein are understood ascomprising enzymes having cellobiohydrolase activity (EC 3.2.1.91),e.g., cellobiohydrolase I and cellobiohydrolase II, as well asendo-glucanase activity (EC 3.2.1.4) and beta-glucosidase activity (EC3.2.1.21).

At least three categories of enzymes are important for convertingcellulose into fermentable sugars: endo-glucanases (EC 3.2.1.4) that cutthe cellulose chains at random; cellobiohydrolases (EC 3.2.1.91) whichcleave cellobiosyl units from the cellulose chain ends andbeta-glucosidases (EC 3.2.1.21) that convert cellobiose and solublecellodextrins into glucose. Among these three categories of enzymesinvolved in the biodegradation of cellulose, cellobiohydrolases seems tobe the key enzymes for degrading native crystalline cellulose.

The cellulolytic activity may, in a preferred embodiment, be in the formof a preparation of enzymes of fungal origin, such as from a strain ofthe genus Trichoderma, preferably a strain of Trichoderma reesei; astrain of the genus Humicola, such as a strain of Humicola insolens; ora strain of Chrysosporium, preferably a strain of Chrysosporiumlucknowense.

In preferred embodiment the cellulolytic enzyme preparation contains oneor more of the following activities: cellulase, hemicellulase,cellulolytic enzyme enhancing activity, beta-glucosidase activity,endoglucanase, cellubiohydrolase, or xylose isomerase.

In a preferred embodiment the cellulase may be a composition as definedin PCT/US2008/065417, which is hereby incorporated by reference. In oneembodiment the cellulolytic enzyme preparation comprising a polypeptidehaving cellulolytic enhancing activity, preferably a family GH61Apolypeptide, preferably the one disclosed in WO 2005/074656 (Novozymes).The cellulolytic enzyme preparation may further comprise abeta-glucosidase, such as a beta-glucosidase derived from a strain ofthe genus Trichoderma, Aspergillus or Penicillium, including the fusionprotein having beta-glucosidase activity disclosed in WO 2008/057637. Ina preferred embodiment the cellulolytic enzyme preparation may alsocomprises a CBH II enzyme, preferably Thielavia terrestriscellobiohydrolase II CEL6A. In another preferred embodiment thecellulolytic enzyme preparation may also comprise cellulolytic enzymes,preferably one derived from Trichoderma reesei or Humicola insolens.

The cellulolytic enzyme preparation may also comprising a polypeptidehaving cellulolytic enhancing activity (GH61A) disclosed in WO2005/074656; a beta-glucosidase (fusion protein disclosed in WO2008/057637) and cellulolytic enzymes derived from Trichoderma reesei.

In an embodiment the cellulolytic enzyme is the commercially availableproduct CELLUCLAST® 1.5L or CELLUZYME™ available from Novozymes A/S,Denmark or ACCELERASE™ 1000 (from Genencor Inc., USA).

A cellulolytic enzyme may be added for hydrolyzing the pre-treatedlignocellulose-containing material. The cellulolytic enzyme may be dosedin the range from 0.1-100 FPU per gram total solids (TS), preferably0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS. In anotherembodiment at least 0.1 mg cellulolytic enzyme per gram total solids(TS), preferably at least 3 mg cellulolytic enzyme per gram TS, such asbetween 5 and 10 mg cellulolytic enzyme(s) per gram TS is(are) used forhydrolysis.

Endoglucanase (EG)

The term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan4-glucanohydrolase (E.C. No. 3.2.1.4), which catalyses endo-hydrolysisof 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives(such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin,beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucansor xyloglucans, and other plant material containing cellulosiccomponents. Endoglucanase activity may be determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

In a preferred embodiment endoglucanases may be derived from a strain ofthe genus Trichoderma, preferably a strain of Trichoderma reesei; astrain of the genus Humicola, such as a strain of Humicola insolens; ora strain of Chrysosporium, preferably a strain of Chrysosporiumlucknowense.

Cellobiohydrolase (CBH)

The term “cellobiohydrolase” means a 1,4-beta-D-glucan cellobiohydrolase(E.C. 3.2.1.91), which catalyzes the hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose, cellooligosaccharides, or any beta-1,4-linkedglucose containing polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain.

Examples of cellobiohydroloses are mentioned above including CBH I andCBH II from Trichoderma reseei; Humicola insolens and CBH II fromThielavia terrestris cellobiohydrolase (CELL6A)

Cellobiohydrolase activity may be determined according to the proceduresdescribed by Lever et al., 1972, Anal. Biochem. 47: 273-279 and by vanTilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh andClaeyssens, 1985, FEBS Letters 187: 283-288. The Lever et al. method issuitable for assessing hydrolysis of cellulose in corn stover and themethod of van Tilbeurgh et al. is suitable for determining thecellobiohydrolase activity on a fluorescent disaccharide derivative.

Beta-Glucosidase

One or more beta-glucosidases may be present during hydrolysis.

The term “beta-glucosidase” means a beta-D-glucoside glucohydrolase(E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducingbeta-D-glucose residues with the release of beta-D-glucose. For purposesof the present invention, beta-glucosidase activity is determinedaccording to the basic procedure described by Venturi et al., 2002, J.Basic Microbiol. 42: 55-66, except different conditions were employed asdescribed herein. One unit of beta-glucosidase activity is defined as1.0 μmole of p-nitrophenol produced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% TWEEN® 20.

In a preferred embodiment the beta-glucosidase is of fungal origin, suchas a strain of the genus Trichoderma, Aspergillus or Penicillium. In apreferred embodiment the beta-glucosidase is a derived from Trichodermareesei, such as the beta-glucosidase encoded by the bgl1 gene (see FIG.1 of EP 562003). In another preferred embodiment the beta-glucosidase isderived from Aspergillus oryzae (recombinantly produced in Aspergillusoryzae according to WO 02/095014), Aspergillus fumigatus (recombinantlyproduced in Aspergillus oryzae according to Example 22 of WO 02/095014)or Aspergillus niger (1981, J. Appl. 3: 157-163). Hemicellulolyticenzymes

According to the invention the pre-treated lignocellulose-containingmaterial may further be subjected to one or more hemicellulolyticenzymes, e.g., one or more hemicellulases.

Hemicellulose can be broken down by hemicellulases and/or acidhydrolysis to release its five and six carbon sugar components.

In an embodiment of the invention the lignocellulose derived materialmay be treated with one or more hemicellulases.

Any hemicellulase suitable for use in hydrolyzing hemicellulose,preferably into xylose, may be used. Preferred hemicellulases includexylanases, arabinofuranosidases, acetyl xylan esterase, feruloylesterase, glucuronidases, endo-galactanase, mannases, endo or exoarabinases, exo-galactanses, and mixtures of two or more thereof.Preferably, the hemicellulase for use in the present invention is anexo-acting hemicellulase, and more preferably, the hemicellulase is anexo-acting hemicellulase which has the ability to hydrolyzehemicellulose under acidic conditions of below pH 7, preferably pH 3-7.An example of hemicellulase suitable for use in the present inventionincludes VISCOZYME™ (available from Novozymes A/S, Denmark).

In an embodiment the hemicellulase is a xylanase. In an embodiment thexylanase may preferably be of microbial origin, such as of fungal origin(e.g., Trichoderma, Meripilus, Humicola, Aspergillus, Fusarium) or froma bacterium (e.g., Bacillus). In a preferred embodiment the xylanase isderived from a filamentous fungus, preferably derived from a strain ofAspergillus, such as Aspergillus aculeatus; or a strain of Humicola,preferably Humicola lanuginosa. The xylanase may preferably be anendo-1,4-beta-xylanase, more preferably an endo-1,4-beta-xylanase ofGH10 or GH11. Examples of commercial xylanases include SHEARZYME™ andBIOFEED WHEAT™ from Novozymes A/S, Denmark.

The hemicellulase may be added in an amount effective to hydrolyzehemicellulose, such as, in amounts from about 0.001 to 0.5 wt. % oftotal solids (TS), more preferably from about 0.05 to 0.5 wt. % of TS.

Xylanases may be added in amounts of 0.001-1.0 g/kg DM (dry matter)substrate, preferably in the amounts of 0.005-0.5 g/kg DM substrate, andmost preferably from 0.05-0.10 g/kg DM substrate.

Xylose Isomerase

Xylose isomerases (D-xylose ketoisomerase) (E.C. 5.3.1.5.) are enzymesthat catalyze the reversible isomerization reaction of D-xylose toD-xylulose. Some xylose isomerases also convert the reversibleisomerization of D-glucose to D-fructose. Therefore, xylose isomarase issometimes referred to as “glucose isomerase.”

A xylose isomerase used in a method or process of the invention may beany enzyme having xylose isomerase activity and may be derived from anysources, preferably bacterial or fungal origin, such as filamentousfungi or yeast. Examples of bacterial xylose isomerases include the onesbelonging to the genera Streptomyces, Actinoplanes, Bacillus andFlavobacterium, and Thermotoga, including T. neapolitana (Vieille etal., 1995, Appl. Environ. Microbiol. 61(5): 1867-1875) and T. maritime.

Examples of fungal xylose isomerases are derived species ofBasidiomycetes.

A preferred xylose isomerase is derived from a strain of yeast genusCandida, preferably a strain of Candida boidinii, especially the Candidaboidinii xylose isomerase disclosed by, e.g., Vongsuvanlert et al.,1988, Agric. Biol. Chem. 52(7): 1817-1824. The xylose isomerase maypreferably be derived from a strain of Candida boidinii (Kloeckera2201), deposited as DSM 70034 and ATCC 48180, disclosed in Ogata et al.,Agric. Biol. Chem. 33: 1519-1520 or Vongsuvanlert et al., 1988, Agric.Biol. Chem. 52(2): 1519-1520.

In one embodiment the xylose isomerase is derived from a strain ofStreptomyces, e.g., derived from a strain of Streptomyces murinus (U.S.Pat. No. 4,687,742); S. flavovirens, S. albus, S. achromogenus, S.echinatus, S. wedmorensis all disclosed in U.S. Pat. No. 3,616,221.Other xylose isomerases are disclosed in U.S. Pat. Nos. 3,622,463,4,351,903, 4,137,126, 3,625,828, HU patent no. 12,415, DE patent2,417,642, JP patent no. 69,28,473, and WO 2004/044129 each incorporatedby reference herein.

The xylose isomerase may be either in immobilized or liquid form. Liquidform is preferred.

Examples of commercially available xylose isomerases include SWEETZYME™T from Novozymes A/S, Denmark.

The xylose isomerase is added to provide an activity level in the rangefrom 0.01-100 IGIU per gram total solids.

Cellulolytic Enhancing Activity

The term “cellulolytic enhancing activity” is defined herein as abiological activity that enhances the hydrolysis of a lignocellulosederived material by proteins having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or in the increase of thetotal of cellobiose and glucose from the hydrolysis of a lignocellulosederived material, e.g., pre-treated lignocellulose-containing materialby cellulolytic protein under the following conditions: 1-50 mg of totalprotein/g of cellulose in PCS (pre-treated corn stover), wherein totalprotein is comprised of 80-99.5% w/w cellulolytic protein/g of cellulosein PCS and 0.5-20% w/w protein of cellulolytic enhancing activity for1-7 day at 50° C. compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS).

The polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a lignocellulose derived material catalyzed by proteinshaving cellulolytic activity by reducing the amount of cellulolyticenzyme required to reach the same degree of hydrolysis preferably atleast 0.1-fold, more at least 0.2-fold, more preferably at least0.3-fold, more preferably at least 0.4-fold, more preferably at least0.5-fold, more preferably at least 1-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, more preferably at least 10-fold, more preferably at least20-fold, even more preferably at least 30-fold, most preferably at least50-fold, and even most preferably at least 100-fold.

In a preferred embodiment the hydrolysis and/or fermentation is carriedout in the presence of a cellulolytic enzyme in combination with apolypeptide having enhancing activity. In a preferred embodiment thepolypeptide having enhancing activity is a family GH61A polypeptide. WO2005/074647 discloses isolated polypeptides having cellulolyticenhancing activity and polynucleotides thereof from Thielaviaterrestris. WO 2005/074656 discloses an isolated polypeptide havingcellulolytic enhancing activity and a polynucleotide thereof fromThermoascus aurantiacus. U.S. Published Application Serial No.2007/0077630 discloses an isolated polypeptide having cellulolyticenhancing activity and a polynucleotide thereof from Trichoderma reesei.

Proteases

A protease may be added during hydrolysis in step ii), fermentation instep iii) or simultaneous hydrolysis and fermentation. The protease maybe any protease. In a preferred embodiment the protease is an acidprotease of microbial origin, preferably of fungal or bacterial origin.An acid fungal protease is preferred, but also other proteases can beused.

Suitable proteases include microbial proteases, such as fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7.

Contemplated acid fungal proteases include fungal proteases derived fromAspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra,Irpex, Penicillium, Scierotiumand Torulopsis. Especially contemplatedare proteases derived from Aspergillus niger (see, e.g., Koaze et al.,1964, Agr. Biol. Chem. Japan 28: 216), Aspergillus saitoi (see, e.g.,Yoshida, 1954, J. Agr. Chem. Soc. Japan 28: 66), Aspergillus awamori(Hayashida et al., 1977, Agric. Biol. Chem. 42(5): 927-933, Aspergillusaculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepAprotease; and acidic proteases from Mucor pusillus or Mucor miehei.

Contemplated are also neutral or alkaline proteases, such as a proteasederived from a strain of Bacillus. A particular protease contemplatedfor the invention is derived from Bacillus amyloliquefaciens and has thesequence obtainable at Swissprot as Accession No. P06832. Alsocontemplated are the proteases having at least 90% identity to aminoacid sequence obtainable at Swissprot as Accession No. P06832 such as atleast 92%, at least 95%, at least 96%, at least 97%, at least 98%, orparticularly at least 99% identity.

Further contemplated are the proteases having at least 90% identity toamino acid sequence disclosed as SEQ. ID. NO:1 in the WO 2003/048353such as at 92%, at least 95%, at least 96%, at least 97%, at least 98%,or particularly at least 99% identity.

Also contemplated are papain-like proteases such as proteases withinE.C. 3.4.22.* (cysteine protease), such as EC 3.4.22.2 (papain), EC3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14(actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycylendopeptidase) and EC 3.4.22.30 (caricain).

In an embodiment the protease is a protease preparation derived from astrain of Aspergillus, such as Aspergillus oryzae. In another embodimentthe protease is derived from a strain of Rhizomucor, preferablyRhizomucor mehei. In another contemplated embodiment the protease is aprotease preparation, preferably a mixture of a proteolytic preparationderived from a strain of Aspergillus, such as Aspergillus oryzae, and aprotease derived from a strain of Rhizomucor, preferably Rhizomucormehei.

Aspartic acid proteases are described in, for example, Handbook ofProteolytic Enzymes, Edited by A. J. Barrett, N. D. Rawlings and J. F.Woessner, Academic Press, San Diego, 1998, Chapter 270. Suitableexamples of aspartic acid protease include, e.g., those disclosed inBerka et al., 1990, Gene 96: 313; Berka et al., 1993, Gene 125: 195-198;and Gomi et al., 1993, Biosci. Biotech. Biochem. 57: 1095-1100, whichare hereby incorporated by reference.

Commercially available products include ALCALASE®, ESPERASE™FLAVOURZYME™, PROMIX™, NEUTRASE®, RENNILASE®, NOVOZYM™ FM 2.0L, andNOVOZYM™ 50006 (available from Novozymes A/S, Denmark) and GC106™ andSPEZYME™ FAN from Genencor Int., Inc., USA.

The protease may be present in an amount of 0.0001-1 mg enzyme proteinper g DS, preferably 0.001 to 0.1 mg enzyme protein per g DS.Alternatively, the protease may be present in an amount of 0.0001 to 1LAPU/g DS, preferably 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/gDS, preferably 0.001 to 0.1 mAU-RH/g DS.

Composition

In this aspect the invention relates to a composition comprising one ormore carbonic anhydrases and one or more carbohydrases. In a preferredembodiment the carbohydrase is an alpha-amylase. In a preferredembodiment the alpha-amylase is an acid amylase or fungal alpha-amylase,preferably an acid fungal alpha-amylase.

In a preferred embodiment the composition comprises enzymes selectedfrom the group consisting cellulolytic enzymes, such as cellulases,and/or hemicellulolytic enzymes, such as hemicellulases. The compositionmay also comprise carbohydrate-source generating enzymes, such asespecially glucoamylases, beta-amylases, maltogenic amylases,pullulanases, alpha-glucosidases, or a mixture thereof.

Examples of contemplated enzymes can be found above in “Enzymes” sectionabove.

Use

In this aspect the invention relates to the use of carbonic anhydrasefor controlling pH fluctuation during fermentation. In a preferredembodiment the fermentation is a process of the invention. The inventionalso relates to the use of carbonic anhydrase for improving yeastfermentation product yields and fermentation rate.

Transgenic Plant Material

In this final aspect the invention relates to transgenic plant materialtransformed with one or more carbonic anhydrase genes.

In another embodiment the invention relates to a transgenic plantcapable of expression one or more carbonic anhydrases in increasedamounts compared to corresponding unmodified plant material.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure, including definitions will becontrolling.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

Materials & Methods Materials:

Carbonic Anhydrase (CA): Carbonic anhydrase derived from Bacillusclausii KSM-K16 (Uniprot acc. No. Q5WD44).Glucoamylase (AMG A): Glucoamylase derived from Trametes cingulatadisclosed in SEQ ID NO: 2 in WO 2006/069289 and available from NovozymesA/S.Alpha-Amylase (AA 1): Hybrid alpha-amylase consisting of Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andSBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S).Yeast: RED STAR™ available from Red Star/Lesaffre, USA

Methods: Identity

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Detection of Carbonic Anhydrase Activity (Wilbur Units)

The test for the detection of carbonic anhydrase was described byWilbur, 1948, J. Biol. Chem. 176: 147-154. The set up is based on the pHchange of the assay mixture due to the formation of bicarbonate fromcarbon dioxide as given in equation 1: [CO₂+H₂O→HCO₃ ⁻+H⁺].

The activity assay used in this study was derived from the procedure ofChirica et al., 2001, Biochim. Biophys. Acta 1544(1-2): 55-63. Asolution containing approximately 60 to 70 mM CO₂ was prepared bybubbling CO₂ into 100 ml distilled water using the tip of a syringeapproximately 45 min to 1 h prior to the assay. The CO₂ solution waschilled in an ice-water bath. To test for the presence of carbonicanhydrase, 2 ml of 25 mM Tris, pH 8.3 (containing sufficient bromothymolblue to give a distinct and visible blue color) were added to two 13×100mm test tubes chilled in an ice bath. To one tube, 10 to 50 μl of theenzyme containing solution (e.g., culture broth or purified enzyme) wasadded, and an equivalent amount of buffer was added to the second tubeto serve as a control. Using a 2 ml syringe and a long cannula, 2 ml ofCO₂ solution was added very quickly and smoothly to the bottom of eachtube. Simultaneously with the addition of the CO₂ solution, a stopwatchwas started. The time required for the solution to change from blue toyellow was recorded (transition point of bromothymol blue is pH 6-7.6).The production of hydrogen ions during the CO₂ hydration reaction lowersthe pH of the solution until the color transition point of thebromothymol blue is reached. The time required for the color change isinversely related to the quantity of carbonic anhydrase present in thesample. The tubes must remain immersed in the ice bath for the durationof the assay for results to be reproducible. Typically, the uncatalyzedreaction (the control) takes approximately 2 min for the color change tooccur, whereas the enzyme catalyzed reaction is complete in 5 to 15 s,depending upon the amount of enzyme added. Detecting the color change issomewhat subjective but the error for triple measurements was in therange of 0 to 1 sec difference for the catalyzed reaction. One unit isdefined after Wilbur [1 U=(1/t_(c))−(1/t_(u))×1000] where U is units andt_(c) and t_(u) represent the time in seconds for the catalyzed anduncatalyzed reactions, respectively (Wilbur, 1948, J. Biol. Chem.176:147-154.

Glucoamylase Activity

Glucoamylase activity may be measured in AGI units or in GlucoamylaseUnits (AGU).

Glucoamylase Activity (AGI)

Glucoamylase (equivalent to amyloglucosidase) converts starch intoglucose. The amount of glucose is determined here by the glucose oxidasemethod for the activity determination. The method described in thesection 76-11 Starch-Glucoamylase Method with Subsequent Measurement ofGlucose with Glucose Oxidase in “Approved methods of the AmericanAssociation of Cereal Chemists”. Vol. 1-2 AACC, from AmericanAssociation of Cereal Chemists, (2000); ISBN: 1-891127-12-8.

One glucoamylase unit (AGI) is the quantity of enzyme which will form 1micro mole of glucose per minute under the standard conditions of themethod.

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch, concentration approx. 16 g dry matter/L.

Buffer: Acetate, approx. 0.04 M, pH=4.3

pH: 4.3

Incubation temperature: 60° C.

Reaction time: 15 minutes

Termination of the reaction: NaOH to a concentration of approximately0.2 g/L (pH-9)

Enzyme concentration: 0.15-0.55 AAU/mL

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as colorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine.

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Acid Alpha-Amylase Activity (AFAU)

When used according to the present invention the activity of an acidalpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units).Alternatively, activity of acid alpha-amylase may be measured in AAU(Acid Alpha-amylase Units).

Acid Alpha-Amylase Units (AAU)

The acid alpha-amylase activity can be measured in AAU (AcidAlpha-amylase Units), which is an absolute method. One Acid Amylase Unit(AAU) is the quantity of enzyme converting 1 g of starch (100% of drymatter) per hour under standardized conditions into a product having atransmission at 620 nm after reaction with an iodine solution of knownstrength equal to the one of a color reference.

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch. Concentration approx. 20 g DS/L.

Buffer: Citrate, approx. 0.13 M, pH=4.2

Iodine solution: 40.176 g potassium iodide+0.088 g iodine/L

City water 15°-20° dH (German degree hardness)

pH: 4.2

Incubation temperature: 30° C.

Reaction time: 11 minutes

Wavelength: 620 nm

Enzyme concentration: 0.13-0.19 AAU/mL

Enzyme working range: 0.13-0.19 AAU/mL

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as colorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine. Further details can befound in EP 0140410 B2, which disclosure is hereby included byreference.

Determination of FAU-F

FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative to anenzyme standard of a declared strength.

Reaction conditions Temperature 37° C. pH 7.15 Wavelength 405 nmReaction time 5 min Measuring time 2 min

A folder (EB-SM-0216.02) describing this standard method in more detailis available on request from Novozymes A/S, Denmark, which folder ishereby included by reference.

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard. 1 AFAU is defined as the amount of enzyme which degrades 5.260mg starch dry matter per hour under the below mentioned standardconditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

Standard Conditions/Reaction Conditions:

-   -   Substrate: Soluble starch, approx. 0.17 g/L    -   Buffer: Citrate, approx. 0.03 M    -   Iodine (12): 0.03 g/L    -   CaCl2: 1.85 mM    -   pH: 2.50±0.05    -   Incubation temperature: 40° C.    -   Reaction time: 23 seconds    -   Wavelength: 590 nm    -   Enzyme concentration: 0.025 AFAU/mL    -   Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Measurement of Cellulase Activity Using Filter Paper Assay (FPUAssay) 1. Source of Method

1.1 The method is disclosed in a document entitled “Measurement ofCellulase Activities” by Adney, B. and Baker, J. 1996. LaboratoryAnalytical Procedure, LAP-006, National Renewable Energy Laboratory(NREL). It is based on the IUPAC method for measuring cellulase activity(Ghose, 1987, Measurement of Cellulase Activities, Pure & Appl. Chem.59: 257-268.

2. Procedure

2.1 The method is carried out as described by Adney and Baker, 1996,supra, except for the use of a 96 well plates to read the absorbancevalues after color development, as described below.

2.2 Enzyme Assay Tubes:

-   2.2.1 A rolled filter paper strip (#1 Whatman; 1×6 cm; 50 mg) is    added to the bottom of a test tube (13×100 mm).-   2.2.2 To the tube is added 1.0 mL of 0.05 M Na-citrate buffer (pH    4.80).-   2.2.3 The tubes containing filter paper and buffer are incubated 5    min. at 50° C. (±0.1° C.) in a circulating water bath.-   2.2.4 Following incubation, 0.5 mL of enzyme dilution in citrate    buffer is added to the tube. Enzyme dilutions are designed to    produce values slightly above and below the target value of 2.0 mg    glucose.-   2.2.5 The tube contents are mixed by gently vortexing for 3 seconds.-   2.2.6 After vortexing, the tubes are incubated for 60 mins. at    50° C. (±0.1° C.) in a circulating water bath.-   2.2.7 Immediately following the 60 min. incubation, the tubes are    removed from the water bath, and 3.0 mL of DNS reagent is added to    each tube to stop the reaction. The tubes are vortexed 3 seconds to    mix.

2.3 Blank and Controls

-   2.3.1 A reagent blank is prepared by adding 1.5 mL of citrate buffer    to a test tube.-   2.3.2 A substrate control is prepared by placing a rolled filter    paper strip into the bottom of a test tube, and adding 1.5 mL of    citrate buffer.-   2.3.3 Enzyme controls are prepared for each enzyme dilution by    mixing 1.0 mL of citrate buffer with 0.5 mL of the appropriate    enzyme dilution.-   2.3.4 The reagent blank, substrate control, and enzyme controls are    assayed in the same manner as the enzyme assay tubes, and done along    with them.

2.4 Glucose Standards

-   2.4.1 A 100 mL stock solution of glucose (10.0 mg/mL) is prepared,    and 5 mL aliquots are frozen. Prior to use, aliquots are thawed and    vortexed to mix.-   2.4.2 Dilutions of the stock solution are made in citrate buffer as    follows:    G1=1.0 mL stock+0.5 mL buffer=6.7 mg/mL=3.3 mg/0.5 mL    G2=0.75 mL stock+0.75 mL buffer=5.0 mg/mL=2.5 mg/0.5 mL    G3=0.5 mL stock+1.0 mL buffer=3.3 mg/mL=1.7 mg/0.5 mL    G4=0.2 mL stock+0.8 mL buffer=2.0 mg/mL=1.0 mg/0.5 mL-   2.4.3 Glucose standard tubes are prepared by adding 0.5 mL of each    dilution to 1.0 mL of citrate buffer.-   2.4.4 The glucose standard tubes are assayed in the same manner as    the enzyme assay tubes, and done along with them.

2.5 Color Development

-   2.5.1 Following the 60 min. incubation and addition of DNS, the    tubes are all boiled together for 5 mins. in a water bath.-   2.5.2 After boiling, they are immediately cooled in an ice/water    bath.-   2.5.3 When cool, the tubes are briefly vortexed, and the pulp is    allowed to settle. Then each tube is diluted by adding 50 microL    from the tube to 200 microL of ddH2O in a 96-well plate. Each well    is mixed, and the absorbance is read at 540 nm.

2.6 Calculations (examples are given in the NREL document)

-   2.6.1 A glucose standard curve is prepared by graphing glucose    concentration (mg/0.5 mL) for the four standards (G1-G4) vs. A₅₄₀.    This is fitted using a linear regression (Prism Software), and the    equation for the line is used to determine the glucose produced for    each of the enzyme assay tubes.-   2.6.2 A plot of glucose produced (mg/0.5 mL) vs. total enzyme    dilution is prepared, with the Y-axis (enzyme dilution) being on a    log scale.-   2.6.3 A line is drawn between the enzyme dilution that produced just    above 2.0 mg glucose and the dilution that produced just below that.    From this line, it is determined the enzyme dilution that would have    produced exactly 2.0 mg of glucose.-   2.6.4 The Filter Paper Units/mL (FPU/mL) are calculated as follows:    FPU/mL=0.37/enzyme dilution producing 2.0 mg glucose

Protease Assay Method—AU(RH)

The proteolytic activity may be determined with denatured hemoglobin assubstrate. In the Anson-Hemoglobin method for the determination ofproteolytic activity denatured hemoglobin is digested, and theundigested hemoglobin is precipitated with trichloroacetic acid (TCA).The amount of TCA soluble product is determined with phenol reagent,which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU-RH) is defined as the amount of enzyme which understandard conditions (i.e., 25° C., pH 5.5 and 10 min. reaction time)digests hemoglobin at an initial rate such that there is liberated perminute an amount of TCA soluble product which gives the same color withphenol reagent as one milliequivalent of tyrosine.

The AU(RH) method is described in EAL-SM-0350 and is available fromNovozymes A/S Denmark on request.

Protease Assay Method (LAPU)

1 Leucine Amino Peptidase Unit (LAPU) is the amount of enzyme whichdecomposes 1 microM substrate per minute at the following conditions: 26mM of L-leucine-p-nitroanilide as substrate, 0.1 M Tris buffer (pH 8.0),37° C., 10 minutes reaction time.

LAPU is described in EB-SM-0298.02/01 available from Novozymes A/SDenmark on request.

Determination of Maltogenic Amylase activity (MANU)

One MANU (Maltogenic Amylase Novo Unit) may be defined as the amount ofenzyme required to release one micro mole of maltose per minute at aconcentration of 10 mg of maltotriose (Sigma M 8378) substrate per ml of0.1 M citrate buffer, pH 5.0 at 37° C. for 30 minutes.

Assay Method for pH Stability of Alpha-Amylase

Alpha-amylase is diluted to appropriate concentration of 0.03 AFAU/ml.The assay is carried out in a micro-plate.

10 microL of diluted enzyme is added to 6 microL of stock buffer (0.5 M)with different pH, ranging from pH 2.5 to 8.0 and added 67 microL ofMilliQ water to final buffer concentration of 0.0375 M. The mixture isincubated at 37° C. for 2 hours. Subsequently the residual activity ofthe enzyme in question is assayed by adding 80 microL of starch workingsolution (final concentration: 0.35 g/l gelatinized starch; 50 mM NaAc,pH 4.0; 0.1 M NaCl; 3 mM CaCl₂). The reaction is carried out at 37° C.for 2 minutes with shaking in the microplate reader and 40 microL offreshly prepared iodine working solution (final concentration: 0.2% KI;0.02% iodine). The mixture is further incubated at 37° C. for 1 minutewithout shaking in the microplate reader. Absorbance at 590 nm is taken.pH stability is determined.

EXAMPLES Example 1 Effect of Carbonic Anhydrase (CA) TowardsAlpha-Amylase (AA 1) and Glucoamylase (AMG A) Combo in One-StepFermentation Process

All treatments were evaluated via mini-scale fermentations. 410 g ofground yellow dent corn (with an average particle size around 0.5 mm)was added to 590 g tap water. This mixture was supplemented with 3.0 ml1 g/L penicillin and 1 g of urea. The pH of this slurry was adjusted to4.5 with 40% H₂SO₄. Dry solid (DS) level was determined to be 35 wt. %.Approximately 5 g of this slurry was added to 20 ml vials. Each vial wasdosed with the appropriate amount of enzyme dosage shown in Table 1below followed by addition of 200 micro liters yeast propagate/5 gslurry (RED STAR™). Actual enzyme dosages were based on the exact weightof corn slurry in each vial. Vials were incubated at 32° C. Ninereplicate fermentations of each treatment were run. Three replicateswere selected for 24 hours, 48 hours and 70 hours time point analysis.Vials were vortexed at 24, 48 and 70 hours and analyzed by HPLC. TheHPLC preparation consisted of stopping the reaction by addition of 50micro liters of 40% H₂SO₄, centrifuging, and filtering through a 0.45micrometer filter. Samples were stored at 4° C. until analysis. Agilent™1100 HPLC system coupled with RI detector was used to determine ethanoland oligosaccharides concentration. The separation column was AminexHPX-87H ion exclusion column (300 mm×7.8 mm) from BioRad™.

TABLE 1 AA 1 dose AMG A dose CA dose Treatments (FAU-F/gDS) (AGU/g DS)(Units/g DS) 1 AA 1 + AMG A 0.0475 0.50 — 2 AA 1 + AMG A + 0.0475 0.50 2,886 CA (X) 3 AA 1 + AMG A + 0.0475 0.50 11,544 CA (Y)Results—Ethanol Yield with Time and Carbonic Anhydrase at DifferentConcentrations

CA (units/g DS) Time (hr) 0 X Y 24 hours 105.23 107.13 108.34 48 hours148.70 151.22 152.66 70 hours 159.79 162.65 163.77

The results are also displayed in FIG. 1.

1. A process of producing a fermentation product, comprising the stepsof: (a) saccharifying a starch-containing material with an alpha-amylaseand a glucoamylase, (b) fermenting with one or more fermenting organismsby adding a carbonic anhydrase before or during fermentation; whereinsteps (a) and (b) are performed simultaneously at a temperature belowthe initial gelatinization temperature of the starch-containingmaterial, and wherein at least one additional enzyme is added duringsaccharifying step (a) and/or fermenting step (b).
 2. The process ofclaim 1, wherein the at least one additional enzyme is selected from thegroup consisting of a glucoamylase, a beta-amylase, a maltogenicamylase, a pullulanase, an alpha-glucosidase, a protease, a xylanase, anarabinofuranosidase, an acetyl xylan esterase, a feruloyl esterase, aglucuronidase, an endo-galactanase, a mannase, an endo- orexo-arabinase, an exo-galactanase, and any combination thereof.
 3. Theprocess of claim 1, wherein at least two additional enzymes are addedduring saccharifying step (a) and/or fermenting step (b).
 4. The processof claim 1, wherein at least three additional enzymes are added duringsaccharifying step (a) and/or fermenting step (b).
 5. The process ofclaim 1, wherein the carbonic anhydrase and/or the at least oneadditional enzyme is expressed in situ by the fermenting organism. 6.The process of claim 5, wherein the fermenting organism is yeast.
 7. Theprocess of claim 6, wherein the yeast is a strain of Saccharomyces orPichia.
 8. The process of claim 7, wherein the yeast is Saccharomycescerevisiae.
 9. The process of claim 1, wherein steps (a) and (b) arecarried out at a temperature in the range of around 25° C. to around 40°C.
 10. The process of claim 1, wherein the carbonic anhydrase isselected from the carbonic anhydrase classes alpha, beta, and gamma. 11.The process of claim 1, wherein the carbonic anhydrase is derived from astrain of Bacillus, Methanobacterium, or Methanosarcina.
 12. The processof claim 1, wherein the carbonic anhydrase is present or added in aconcentration of 100-100,000 Units/g dry solids.
 13. The process ofclaim 1, wherein the dry solid content (DS) is in the range from 10-55wt. %.
 14. The process of claim 1, wherein the sugar concentration ismaintained at a level below about 6 wt. % during saccharification andfermentation.
 15. The process of claim 1, wherein the carbonic anhydraseis added to the fermentation medium simultaneous with or after analpha-amylase or carbohydrate-source generating enzyme is added.
 16. Theprocess of claim 1, wherein the starch-containing material is selectedfrom the group consisting of corn, cassava, wheat, barley, rye, milo andpotatoes; or any combination thereof.
 17. The process of claim 1,wherein the starch-containing material is corn.
 18. The process of claim1, wherein the fermentation product is ethanol.
 19. The process of claim1, further comprising, prior to steps (a) and (b), the steps of: x)reducing the particle size of starch-containing material; and y) forminga slurry comprising the starch-containing material and water.
 20. Theprocess of claim 1, wherein the saccharification is carried out using acarbohydrate-source generating enzyme selected from the group consistingof beta-amylase, maltogenic amylase, pullulanase, and alpha-glucosidase;or a mixture thereof.